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Relationship between sperm DNA damage, induced acrosome reaction and viability in ICSI patients
Abstract
The DNA damage in human spermatozoa is a relevant predictor of prognosis in male infertility, whereby increased sperm DNA damage impairs the outcomes of artificial reproduction. Theoretically, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Nevertheless, intracytoplasmic sperm injection (ICSI) has been reported to alleviate such negative outcomes due to DNA damage. This study investigated the relationship between DNA fragmentation and acrosome reaction as well as viability in ICSI patients. The study enrolled 42 men undergoing ICSI due to poor sperm parameters. The DNA fragmentation indexes (DFI) were 4-10% in 38% of the cases, and > or = 10% in 19% of the cases. The results of both acrosome reaction and viability assays showed negative correlations with DFI values in all cases and especially in cases with fertilization rates <60% (P < 0.05). However, such correlations were not found in cases with fertilization rates >60%. There were no live deliveries in patients with high DFI levels (>10%). In conclusion, negative correlations were identified between increased DNA damage, and acrosome reaction and/or viability of human spermatozoa, especially in cases with reduced fertilization rates.
... Recent studies suggest that sperm DNA integrity may be altered by environmental exposure to some toxic chemicals (Aitken RJ, De Luiis GN., 2010). DNA fragmentation may be an excellent marker for exposure to potential reproductive toxicants and a diagnostic tool for male infertility (Ozmen B, et al., 2007). The aim of this study was to investigate the association between sperm DNA fragmentation in the population that lives in an environment of air pollution and exposure to pesticides. ...
... Recent studies suggest that sperm DNA integrity may be altered by environmental exposure to some toxic chemicals (Aitken RJ, De Luiis GN., 2010). DNA fragmentation may be an excellent marker for exposure to potential reproductive toxicants and a diagnostic tool for male infertility (Ozmen B, et al., 2007). ...
... that 30 spermatogonial stem cell divisions take place before puberty, when they begin to undergo meiotic divisions. From then on,23 mitotic divisions per year occur, resulting in 150 replications by the age of 20 years and 840 replications by the age of 50 years (Crow JF., 2000). Because of these numerous divisions of stem cells, men may have an increased risk of errors in DNA transcription. ...
Recent studies suggest that sperm DNA integrity may be altered by environmental exposure to some toxic chemicals (Aitken RJ, De Luiis GN., 2010). DNA fragmentation may be an excellent marker for exposure to potential reproductive toxicants and a diagnostic tool for male infertility (Ozmen B, et al., 2007). The aim of this study was to investigate the association between sperm DNA fragmentation in the population that lives in an environment of air pollution and exposure to pesticides. Patients and selection. Total 164 samples of semen from fertile men with respect to the parameters of semen and DNA fragmentation. This is a prospective study observation spent on Andrology Laboratory Polyclinic,, Biolab-Zafi,, in Klina in the Republic of Kosovo from April 2015 to Februare 2016. A portion of each semen sample was used to analyze the following parameters according to the World Health Organization (WHO) guidelines (2010). Halosperm is the SCD test based on the ability of intact DNA with deproteinized nuclei to create loops around nuclear matrix. Deproteinized nuclei create the halos of dispersed DNA that correspond to relaxed DNA loops attached to the residual nuclear structure (core) (Fernández et al., 2005). Results. The group of respondents farmers (exposed to pesticides) that have been tested in the period spring-summer, have a higher percentage index fragmented sperm (DFI = 36.2 ± 4.85), in contrast to the other two groups; Group 2 (air pollution), DFI = 27.8 ± 1.3, and the Group 1 (normal control), DFI = 18.5 ± 6.5 (** P <0.05 Groups 1 and 2). Elezaj et al. World Journal of Pharmaceutical Research and conclusions. Exposure to pesticides increases the percentage of DNA-fragmented sperm in normospermic men ejaculate. To reduce the percentage of DNA-fragmented sperm in the ejaculate, especially in infertile men, should be taken into account and avoid exposure to pesticides by taking antioxidant therapy.
... In effect, the lack of an effective sperm selection method in vitro for use in assisted reproductive technologies (ARTs) is thought to be one of the reasons for the relative low pregnancy rates achieved by procedures such as intracytoplasmic sperm injection (ICSI) in human clinical practice [10][11][12] . Hence, developing an in vitro sperm selection method that will effectively exclude spermatozoa with damaged DNA is one of the main challenges of ARTs, especially of ICSI 1,[12][13][14] . ...
... tract. This could explain the low efficiency of both human [10][11][12][13][14] and animal 37,54-56 infertility treatments. Currently, ICSI is substituting IVF as the first option in human fertility treatment even when no male factor is present 19,57 . ...
... Currently, ICSI is substituting IVF as the first option in human fertility treatment even when no male factor is present 19,57 . Given that infertile men subjected to fertility treatments show higher percentages of spermatozoa with fragmented DNA 13,58,59 , and that sperm DNA damage negatively affects clinical pregnancy following ART 60 , it has been stressed that the development of more selective methods for separating undamaged spermatozoa bearing high integrity DNA is essential to ensure the health of ART-derived individuals 1 . This is especially critical in the case of ICSI whereby all selective barriers for spermatozoa are bypassed, including the later barriers present in IVF such as cumulus penetration, membrane recognition and membrane fusion. ...
The ejaculate is a heterogeneous pool of spermatozoa containing only a small physiologically adequate subpopulation for fertilization. As there is no method to isolate this subpopulation, its specific characteristics are unknown. This is one of the main reasons why we lack effective tools to identify male infertility and for the low efficiency of assisted reproductive technologies. The aim of this study was to improve ICSI outcome by sperm selection through thermotaxis. Here we show that a specific subpopulation of mouse and human spermatozoa can be selected in vitro by thermotaxis and that this subpopulation is the one that enters the fallopian tube in mice. Further, we confirm that these selected spermatozoa in mice and humans show a much higher DNA integrity and lower chromatin compaction than unselected sperm, and in mice, they give rise to more and better embryos through intracytoplasmic sperm injection, doubling the number of successful pregnancies. Collectively, our results indicate that a high quality sperm subpopulation is selected in vitro by thermotaxis and that this subpopulation is also selected in vivo within the fallopian tube possibly by thermotaxis.
... A strong negative relationship was observed between sperm DNA damage analysed by TUNEL and FCCE assays and sperm concentration, progressive motility and normal morphology. The current literature shows mixed data, with some studies showing good association and others finding no correlations (Greco et al., 2005;Nicopoullos et al., 2005;Zini et al., 2005;Ozmen et al., 2007;Lin et al., 2008;Nijs et al., 2009;Tavalaee et al., 2009). Such controversies may arise due to variability in laboratory conditions, lack of standard protocols for DNA damage assays and inter-and intra-individual variability in the test parameters. ...
... This is consistent with our earlier results in which we found that sperm DNA damage affected early embryo development, and in patients with low sperm DNA damage .50% of the embryos developed into blastocysts. These results indicate that sperm DNA damage may affect implantation rates and be associated with rates of spontaneous pregnancy loss (Benchaib et al., 2007;Ozmen et al., 2007;Frydman et al., 2008;Lin et al., 2008), a phenomenon termed 'late paternal effect' by Tesarik et al. (2004). Therefore, we could speculate that decision-making in ART could be adjusted based on sperm DNA damage to reduce the chances of multiple births. ...
... This value also corresponds to the highest OR to obtain a successful clinical pregnancy. A 10% threshold value was calculated for the TUNEL assay, which was previously reported by Borini et al. (2006) and Ozmen et al. (2007). ...
Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)?
Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study.
Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown.
Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013.
Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated.
Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2 ratio was associated with sperm count (P = 0.013), men's age (P = 0.037), maturity (P = 0.049) and blastocyst quality (P = 0.012). Histone retention and sperm DNA damage measured by Comet and TUNEL assays were associated with fertilization rate (P < 0.05), embryo quality (P < 0.05) and implantation rate (P < 0.05).
A potential drawback of this study is that it is cross-sectional. Generally in such studies there is more than one variable that could cause the effect. Analysing sperm is one part of the equation; there are also a number of female factors that have the potential to influence ART outcomes. Therefore, given the large and well-established role of female factors in infertility, normal sperm DNA integrity and protamination do not necessarily ensure clinical pregnancy in ART. Thus, female factors can reduce the prognostic value of sperm DNA tests. Further, our use of native semen instead of prepared sperm may have iatrogenically increased the DNA damage.
Alteration in sperm nuclear protein affects sperm DNA integrity. Further, with the current dataset, TUNEL and Comet assays appeared more predictive of ART success than FCCE.
No personal or direct financial support has been received for any of this work. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.
... Unfortunately, as in ICSI there is no selection based on DNA integrity, it is possible that functionally abnormal spermatozoa are injected. Spermatozoa with fragmented DNA are known to be associated with lower fertilization rates (Ozmen et al., 2007), embryonic development arrest, low implantation and pregnancy rates (Benchaib et al., 2007;Virro et al., 2004), increased incidence of spontaneous abortion (Dar et al., 2013), lower rates of live births (Simon et al., 2013) and/or high morbidity in offspring originated by ICSI (Fernández-Gonzalez et al., 2008). Thus, the sperm DNA integrity may be considered of extreme importance for obtaining a pregnancy (Agarwal & Allamaneni, 2005). ...
... Sperm DNA fragmentation has been considered as an important factor that may negatively affect ICSI outcomes (Benchaib et al., 2007;Dar et al., 2013;Ozmen et al., 2007;Simon et al., 2013;Virro et al., 2004). However, the widely utilized methods for analyzing sDNAfrag preclude using the evaluated sperm in ART (Sharma et al., 2021). ...
Objective:
Despite higher sperm DNA fragmentation may affect intracytoplasmic sperm injection (ICSI) outcomes, sperm selection protocols do not evaluate this parameter. Therefore, sperm's head birefringence has been suggested as an adjuvant of seminal processing to select viable sperm for couples with severe male factor. Considering men with normal seminal parameters may also curse with DNA fragmentation, the aim of this study was to evaluate the impact of sperm selection by birefringence on ICSI outcomes in couples with different infertility factors compared to those submitted to conventional sperm selection.
Methods:
In this case-control study, medical records from 181 couples who underwent ICSI from January 2018 to August 2020 (107 from the Conventional and 74 from the Birefringence group) were included in the study. Clinical characteristics and ICSI outcomes were compared between the groups using Student's t test or Chi-square test (p<0.05) and a multivariate logistic regression model was applied regarding clinical pregnancy.
Results:
Despite the Birefringence group showed higher female age (p=0.01), lower seminal sperm concentration (p<0.01) and higher sperm DNA fragmentation (p<0.01), those patients cursed with both higher cleavage rate (p=0.04), clinical pregnancy rate per transfer (p=0.03) and clinical pregnancy rate per initiated cycle (p=0.02). The logistic regression showed a positive group effect on clinical pregnancy.
Conclusions:
The findings suggest a positive clinical impact of this cheap and easily reproducible adjuvant technique on ICSI outcomes in couples with different infertility factors. If confirmed by further methodologically appropriate studies, the sperm's head birefringence could be considered to improve the reproductive chances of those patients.
... cluded(Aguilera Duvisón et al., 2008;Check et al., 2005;Esteves et al., 2015;Henkel et al., 2003;Ozmen et al., 2007; Alvarez Sedo et al., 2017;Tandara et al., 2013). ...
... Second, 211 irrelevant studies, 103 non-comparative studies, 19 animal model studies and four studies for which the full text could not be found were excluded. Third, 284 meeting abstracts, 86 reviews or guidelines, seven letters or comments, four duplicate report studies and 50 studies for which data could not be extracted were excluded.Fourth, seven studies scoring 5 on the modified Newcastle-Ottawa scale were excluded because of its inclusion of unrepresentative participants and not control baseline factors(Aguilera Duvisón et al., 2008;Check, Graziano, Cohen, Krotec, & Check, 2005;Esteves, Sanchez-Martin, Sanchez-Martin, Schneider, & Gosalvez, 2015;Henkel et al., 2003;Ozmen et al., 2007; Alvarez Sedo et al., 2017;Tandara et al., 2013) In addition, six articles(Cebesoy, Ünlü, Aydos, & Baltaci, 2006;Dar et al., 2013;Jiang, He, Wang, & Zhu, 2011;Wang et al., 2012;Zhang et al., 2008;Zini et al., 2005) measuring DNA fragmentation by the acridine orange test were excluded. Six articles(Belloc et al., 2008;Cebesoy et al., 2006;Duran, Morshedi, Taylor, & Oehninger, 2002;Hu, Zhu, Liu, & Fan, 2011;Muriel et al., 2006;Ren, Sun, Ku, Chen, & Wu, 2004;Yang et al., 2011) in which couples received intrauterine insemination (IUI) were excluded. ...
Studies have explored the influence of DNA damage in assisted reproductive technology (ART), but the outcome remains controversial. To determine whether sperm DNA fragmentation index (DFI) has any effect on ART outcomes, we collected detailed data regarding 1,333 IVF cycles performed at our centre, and the data of our retrospective cohort study were extracted for this meta‐analysis. We searched PubMed, Web of Science, EMBASE and Google Scholar and performed a systemic review and meta‐analysis. Primary meta‐analysis of 10 studies comprising 1,785 couples showed that live birth rate was no significantly different between low‐DFI group and high‐DFI group (p > 0.05). Secondary meta‐analysis of 25 studies comprising 3,992 couples showed a higher miscarriage rate in high‐DFI group than in low‐DFI group (RR=1.57 [1.18, 2.09], p < 0.01). Meta‐analysis of eight studies comprising 17,879 embryos revealed a lower good‐quality embryo rate (RR=0.65 [0.62, 0.68], p < 0.01). Meta‐analysis of 23 studies comprising 6,771 cycles showed that the high‐DFI group had a lower clinical pregnancy rate than low‐DFI group (RR=0.85 [0.75, 0.96], p < 0.01). Heterogeneity of included studies weakened our conclusions. Our study showed that DFI has adverse effects on ART outcome. More well‐designed studies exploring the association between DFI and ART outcome are desired.
... It has also reported that induction of the acrosome reaction in human spermatozoa is associated with improved fertilization and embryo development (Lee et al., 1997;Mansour et al., 2008;Sathananthan et al., 1997). On the other hand, negative correlations between increased DNA damage and acrosome reaction have been identified (Ozmen et al., 2007). With this in mind, the objective of this study was to investigate whether there was any relationship between DNA damage (fragmentation and denaturation) and two types of SHBF: total and partial. ...
... However, DNA damage should alter the special cellular functions of human spermatozoa and lead to diminished acrosome reaction with reduced fertilization rates. Moreover, negative correlation was identified between DNA damage and acrosome reaction and/or viability of human spermatozoa (Ozmen et al., 2007). ...
... However, in vitro manipulations of sperm, such as processing and cryopreservation [1][2][3][4][5][6][7][8][9] can harm sperm DNA integrity and increase the DNA fragmentation index (DFI). Several studies have linked abnormal DFI with decreased success rates after ART [10][11][12], impaired embryo development, lower chances of achieving a pregnancy [13], higher miscarriage rates [14,15], and potentially severe defects in the offspring [16]. ...
Objective
To evaluate whether the ZyMōt™ Multi 850 μl sperm separation device (SSD) effectively recovers motile spermatozoa from cryopreserved ejaculates and compare its effect on key embryology outcomes including fertilization, cleavage stage, and total and top-quality blastocyst formation rates to the traditional Density Gradient Centrifugation (DGC) method.
Methods
In this prospective, single-center, controlled study, we used fresh sibling donor oocytes and non-donor cryopreserved ejaculates. In total, 150 couples participated in this study. At least eight MII donor oocytes were allocated to each couple split into two arms. One arm underwent ICSI with the control DGC-processed sample, and the other arm processed with SSD.
Results
No significant difference on fertilization and cleavage stage embryo rates was observed between the two techniques. We observed a significant increase in the percentage of total (SSD: 74.03 ± 23.47% vs. DGC: 67.86 ± 23.92%; p = 0.016) and top-quality (SSD: 66.38 ± 24.94% vs. DGC: 60.98 ± 24.40%; p = 0.035) blastocysts formed post-SSD processing. Sub-analysis showed that this increase remained significant for the WHO-normal group (n = 118), but not for the WHO-abnormal group (n = 32).
Conclusion
The SSD was successfully applied in all 150 cases, providing adequate numbers of spermatozoa to undergo ICSI. Additionally, SSD significantly improved blastocyst development rates; however, this was of limited clinical impact considering the minor improvement on the average number of top-quality blastocysts. It can be hypothesized that this positive contribution may be stronger and clinically significant when a larger number of oocytes is used or in homologous oocyte ICSI cycles, where the repair mechanisms of the oocytes may insufficient for promoting healthy embryo development.
... In the present work, neither sperm viability nor ROS generation were affected by incubation with Mn 2+ /Ca 2+ or Mg 2+ /Ca 2+ . In mammals, sperm DNA fragmentation is usually correlated with oxidative stress, and ROS is understood to be the main source of DNA damage [52][53][54][55]. The fact that, even when SCF was induced with Mn 2+ /Ca 2+ or Mg 2+ /Ca 2+ , ROS levels were low supports that an enzymatic, non-oxidative mechanism would be activated in this case. ...
Background
In vitro incubation of epididymal and vas deferens sperm with Mn ²⁺ induces Sperm Chromatin Fragmentation (SCF), a mechanism that causes double-stranded breaks in toroid-linker regions (TLRs). Whether this mechanism, thought to require the participation of topoisomerases and/or DNAses and thus far only described in epididymal mouse sperm, can be triggered in ejaculated sperm is yet to be elucidated. The current study aimed to determine if exposure of pig ejaculated sperm to divalent ions (Mn ²⁺ and Mg ²⁺ ) activates SCF, and whether this has any impact on sperm function and survival. For this purpose, sperm DNA integrity was evaluated through the Comet assay and Pulsed Field Gel Electrophoresis (PFGE); sperm motility and agglutination were assessed with computer assisted sperm analysis (CASA); and sperm viability and levels of total reactive oxygen species (ROS) and superoxides were determined through flow cytometry.
Results
Incubation with Mn ²⁺ /Ca ²⁺ activated SCF in a dose-dependent ( P < 0.05) albeit not time-dependent manner ( P > 0.05); in contrast, Mg ²⁺ /Ca ²⁺ only triggered SCF at high concentrations (50 mM). The PFGE revealed that, when activated by Mn ²⁺ /Ca ²⁺ or Mg ²⁺ /Ca ²⁺ , SCF generated DNA fragments of 33–194 Kb, compatible with the size of one or multiple toroids. Besides, Mn ²⁺ /Ca ²⁺ affected sperm motility in a dose-dependent manner ( P < 0.05), whereas Mg ²⁺ /Ca ²⁺ only impaired this variable at high concentrations ( P < 0.05). While this effect on motility was concomitant with an increase of agglutination, neither viability nor ROS levels were affected by Mn ²⁺ /Ca ²⁺ or Mg ²⁺ /Ca ²⁺ treatments.
Conclusion
Mn ²⁺ /Ca ²⁺ and Mn ²⁺ /Ca ²⁺ were observed to induce SCF in ejaculated sperm, resulting in DNA cleavage at TLRs. The activation of this mechanism by an intracellular, non-oxidative factor sheds light on the events taking place during sperm cell death.
... This result was consistent with the highly positive correlation (0.89) between MM and NC as shown in Table 5. In this regard, it has been established that sperm morphological abnormalities are related to chromatin integrity loss (Tavalaee et al., 2009), progressive motility and viability loss (Ozmen et al., 2007). The percentage of live-reacted spermatozoa (LR) for the three ram groups were 52.73%, 45.58% and 34.71% for high, medium and low fertility ram groups, respectively, with significant (P<0.05) ...
... Oxidative damage is increased in samples with lower sperm motility [58], while motility presents an inverse relationship with DNA fragmentation [59,60]. Higher levels of DNA fragmentation, which may derive from oxidative stress, result in poorer ART outcomes [61,62] and may contribute to recurrent pregnancy failures and increased incidence of miscarriage [63,64]. In addition, metagenomic studies have demonstrated that the seminal microbiome is linked to alterations in sperm motility and an increase in DNA fragmentation as a result of inflammatory cytokines, overproduction of ROS, direct interaction of pathogens with sperm, or indirectly through the secretion of soluble factors [65,66]. ...
This retrospective cohort study aimed to explore whether paternal age and semen quality parameters affect the embryological and clinical outcomes of ICSI with oocyte donation. A total of 339 oocyte donation (OD)-ICSI cycles were categorized into four groups according to the semen parameter profiles of the male counterparts: normozoospermia (NS, n = 184), oligozoospermia (OS, n = 41), asthenozoospermia (AS, n = 50), and oligoasthenozoospermia (OAS, n = 64). The effect of age, total sperm count, and progressive motility was separately analyzed for reproductive outcomes and compared between the study groups: fertilization, blastulation, and top-quality embryo rate, biochemical and clinical pregnancy, live birth, and miscarriage. A negative correlation between male age and fertilization rate was observed (rs = − 0.23, p < 0.0001), while male age was a significant factor for biochemical pregnancy (p = 0.0002), clinical pregnancy (p = 0.0017), and live birth (p = 0.0038). Reduced total sperm count and lowered progressive motility led to poorer fertilization rates (rs = 0.19 and 0.35, respectively, p < 0.0001) and affected embryo quality (rs = 0.13, p = 0.02, and rs = 0.22, p < 0.0001, respectively). OD-ICSI cycles with asthenozoospermia had significantly lowered success rates in biochemical pregnancy, clinical pregnancy, and live birth (p < 0.05). Our study demonstrated that both advanced male age and reduced progressive motility of spermatozoa exert a significant negative influence on the outcome of assisted reproduction, even in controlled procedures with gamete selection and optimization such as in OD-ICSI. Improvement in treatment strategies and male fertility evaluation requires incorporation of such evidence to obtain better prognosis towards personalized management.
... Semen fertility using a combination of laboratory tests to predict the characteristics of different sperm. It has been documented that loss of chromatin integrity is associated with sperm morphological abnormalities (Tavalaee et al., 2009), loss of viability and progressive motility (Ozmen et al., 2007), and decreased concentration and maturation of sperm (Virro et al., 2004). ...
... Semen fertility using a combination of laboratory tests to predict the characteristics of different sperm. It has been documented that loss of chromatin integrity is associated with sperm morphological abnormalities (Tavalaee et al., 2009), loss of viability and progressive motility (Ozmen et al., 2007), and decreased concentration and maturation of sperm (Virro et al., 2004). ...
The aim of this study was to determine goat breed (Zaraibi and Baladi) or buck age (young and old) effect on characteristics, production, flow cytometer, DNA fragmentation and comet assay parameters of spermatozoa. Twelve Baladi (BG) and Zaraibi (ZG) bucks, 3 old (2-4 years) and 3 young (8-12 months) from BG or ZG used for semen collection for 24 weeks. Volume (EV), sperm progressive motility (PM), livability (LS), abnormality (SA), and concentration (SCC) were evaluated, sperm outputs (SO) were calculated. Sperm apoptosis, DNA fragmentation and comet assay were analyzed. Results revealed that PM, LS, SA, SCC, total, motile, live, normal, and functional SO per ejaculate were significantly higher in ZG than BG and in old than young bucks in each breed. Both EV and LS were significantly higher, while SCC was significantly lower in old than young ZG. Each of EV, PM, and SCC was significantly higher, while SA was significantly lower in old than young BG. Viable sperm, haploid, and cell cycle percent were significantly higher, while early apoptotic, apoptotic, and necrotic spermatozoa percent was significantly lower in old than young bucks of each breed. Spermatid percent in ZG and diploid percent in BG were significantly higher in old than young bucks. Tailed sperm, tail length, tail DNA percentage, and tail moment were significantly lower in ZG than BG, and in old than young bucks of each breed. It can be stated that Zaraibi bucks have a good potential for semen production than Baladi breed.
... sperm travel in the female reproductive tract, establishment of oviductal sperm reservoir and fertilization of oocyte. We observed that the proportion of spermatozoa with compromised membrane and acrosome was higher in bulls with high sperm DFI%, which is in agreement with earlier reports [11,34,42,43] who also observed a negative relationship of high-DFI% with viable and acrosome reacted spermatozoa. Spermatozoa with damaged DNA seem to decrease the viable proportion of the spermatozoa, which may occur either through apoptotic endonuclease activation or through necrosis as described in humans [11]. ...
In the present study, we standardized an in vitro oviduct explants model for cattle and assessed the oviduct explants binding ability and phenotypic characteristics of spermatozoa obtained from breeding bulls with high- and low-sperm DNA fragmentation index (DFI%). Cryopreserved spermatozoa from Holstein Friesian crossbred breeding bulls (n=45) with known field fertility were assessed for DFI% and were classified into either high DFI% or low DFI% category. Flow cytometry was used to assess sperm membrane integrity, acrosome reaction status, mitochondrial membrane potential and intracellular calcium concentrations. It was found that spermatozoa from bulls with low DFI% had significantly higher (P<0.05) membrane integrity, acrosome intactness, and mitochondrial membrane potential. To assess the sperm oviduct binding ability, oviduct explants were prepared by incubating the oviduct cells overnight in TCM-199 medium at 38.5°C under 5% CO2. Different sperm concentrations and times of incubation were evaluated and found that 2 million spermatozoa and 1-hour incubation yielded high binding index (BI). The BI was also significantly (P<0.01) higher (>2 times) in the bulls with low-DFI% as compared to high DFI% bulls. The correlation between binding index and DFI% was negative and significant (r = -.528; P<0.05). Further, the binding index was positively correlated with conception rate (r = .703), intact sperm membrane (r = .631) and mitochondrial membrane potential (r = .609). It is inferred that sperm phenotypic characteristics and oviduct binding ability are impaired in breeding bulls with high sperm DFI%, which might be associated with low conception rates in these bulls.
... This is especially relevant in case of the intracytoplasmic sperm injection (ICSI) and could explain the low efficiency of ART in general [28]. Nearly half of the male patients diagnosed as infertile show high levels of sperm DNA damage [29], and most patients subjected to fertility treatments show alterations in the sperm chromatin [30][31][32][33]. Moreover, low sperm counts have been related to higher presence of chromosomal aberrations in the spermatozoa. ...
... Phản ứng acrosome và xét nghiệm sức sống tinh trùng có mối tương quan với giá trị đứt gãy DNA, đặc biệt trong trường hợp tỷ lệ thụ tinh <60%. Có thể thấy rằng sức sống tinh trùng có mối tương quan đến tổn thương DNA tinh trùng [15]. ...
Đặt vấn đề: Kiểm tra sức sống tinh trùng (sperm survival test – SST) được phát triển như là một xét nghiệm nâng cao để đánh giá khả năng sống của tinh trùng trong điều kiện in vitro. Chúng tôi tiến hành đánh giá sức sống của tinh trùng sau lọc rửa với các mốc thời gian 0 giờ, 24 giờ và 48 giờ từ đó nghiên cứu ảnh hưởng của sức sống tinh trùng đến kết quả ICSI. Đối tượng và phương pháp nghiên cứu: Nghiên cứu hồi cứu trên 100 cặp vợ chồng vô sinh điều trị tại Trung tâm Nội tiết Sinh sản và Vô sinh – Bệnh viện Đại học Y Dược Huế (HUECREI) từ tháng 01/2017 đến tháng 12/2017. Mẫu tinh trùng được lọc rửa vả nuôi cấy. Độ di động, sức sống được đánh giá sau khi nuôi cấy cùng với kết quả ICSI. Kết quả: Nhóm có sức sống tinh trùng bình thường (tỷ lệ tinh trùng sống ≥58% sau 24 giờ và ≥20% sau 48 giờ nuôi cấy) – nhóm 1 cho MSI (motility survival index) và VSI (vitality survival index) cao hơn nhóm 2 – nhóm có sức sống tinh trùng bất thường, lần lượt là 55,7% so với 26,72% và 77,83% so với 41,47% (p<0,05). Tỷ lệ thụ tinh và tỷ lệ tạo phôi tốt của nhóm 1 (85,29% và 71,38%) cao hơn nhóm 2 (81,74% và 50,15%). Nhóm 1 có tỷ lệ thai lâm sàng và tỷ lệ thai diễn tiến (lần lượt là 38,18% và 29,1%) khác biệt so với nhóm 2 (lần lượt là 24,44% và 28,89%). Kết luận: Tỷ lệ tinh trùng di động tiến tới và sức sống tinh trùng giảm dần theo thời gian nuôi cấy. Sức sống tinh trùng có mối tương quan đến kết quả thụ tinh trong ống nghiệm: kết quả thụ tinh trong ống nghiệm tốt hơn khi sử dụng tinh trùng có sức sống bình thường, nhóm sử dụng tinh trùng có sức sống bình thường có tỷ lệ thụ tinh, tỷ lệ tạo phôi tốt, tỷ lệ thai lâm sàng và tỷ lệ thai diễn tiến cao (lần lượt là 85,29%, 71,38%, 38,18% và 29,1%).
... Furthermore, studies of Arpanahi et al. (95) and Hammound et al. (96) highlighted the significant role of retained histones in early embryonic development, zygotic genome activation, signaling pathways and imprinting genes, in the case of human and rat spermatozoa. In addition, it is documented that loss of chromatin integrity is associated with sperm morphological abnormalities (97), loss of viability and progressive motility (98), reduced concentration (45) and sperm maturity (99). Furthermore, Carrel and Liu (100) and Virro et al. (99) depicted a strong association between loss of chromatin integrity and poor implantation or spontaneous abortion. ...
The accurate prediction of male fertility is of major economic importance in the animal breeding industry. However, the results of conventional semen analysis do not always correlate with field fertility outcomes. There is evidence to indicate that mammalian fertilization and subsequent embryo development depend, in part, on the inherent integrity of the sperm DNA. Understanding the complex packaging of mammalian sperm chromatin and assessment of DNA integrity could potentially provide a benchmark in clinical infertility. In the era of assisted reproduction, especially when in-vitro fertilization or gamete intrafallopian transfer or intracytoplasmic sperm injection is used, assessment of sperm DNA integrity is important because spermatozoa are not subjected to the selection process occurring naturally in the female reproductive tract. Although sperm DNA integrity testing measures a significant biological parameter, its precise role in the infertility evaluation in farm animals remains unclear. In this review, the earlier findings on sperm DNA integrity in relation to male fertility are compiled and analyzed. Furthermore, the causes and consequences of sperm DNA damage are described, together with a review of advances in methods for detection of sperm DNA damage, and the prognostic value of sperm DNA quality on male fertility.
... Una amplia serie de estudios que han determinado la correlación entre el daño del ADN espermático y los parámetros seminales en diferentes poblaciones y utilizando diferentes ensayos (12-23), sin embargo también existen estudios que no han determinado ninguna correlación (24,25). Así mismo un alto índice de fragmentación del ADN espermático se ha relacionado negativamente con la fertilidad, con el desarrollo embrionario (26) y con una baja tasa de formación de blastocistos y de embarazo tras la fertilización In Vitro (FIV) e inyección intracitoplasmática del espermatozoide (ICSI) (27)(28)(29)(30)(31)(32)(33) (34)(35)(36)(37)(38)(39)(40). Halosperm es un test SCD nuevo, mejorado, económico y simple que se basa en la descondensación diferencial de la cromatina en aquellos espermatozoides que tienen su ADN fragmentado respecto aquellos que no lo tienen. ...
RESUMEN
Objetivo: Determinar la correlación entre los parámetros seminales (concentración, movilidad, morfología y vitalidad) y la integridad del ADN espermático, utilizando el test Halosperm. Material y métodos:Estudio prospectivo realizado en el Laboratorio de Andrología del Laboratorio de Reproducción Asistida FERTILAB, Lima – Perú, desde el mes de Agosto del 2012 al mes de Marzo del 2013. Se evaluaron 282 pacientes. Se determinó el Índice de Fragmentación de ADN (IFA) espermático correspondiente a los pacientes con muestra seminal normozoospermica y pacientes con muestra seminal alterado en algún parámetro seminal. Se utilizaron dos puntos de corte de IFA (18% y 30%) y se correlacionaron con los parámetros seminales. En la población total se determinó la correlación de Spearman entre edad, los parámetros seminales y el IFA. Resultados:Se determinó que el valor de la mediana de IFA de los varones con muestra seminal normozoospermica era significativamente menor que el de los varones con muestra seminal alterado en algún parámetro (12,8% vs 19,0, P=0,000). Utilizando el punto de corte de 18% se encontró que existe diferencia significativa en las medianas entre los dos grupos (Grupo 1: ≤ 18% y Grupo 2: > 18%) en la edad (37años vs 40 años, P=0,002), concentración espermática (82,30 x 106/ml vs 58,00 x 106/ml, P=0,046), movilidad progresiva (45,80% vs 27,40, P=0,000), morfología normal (12,50% vs 9,00%, P=0,000) y vitalidad espermática (89,0% vs 78,0%, P=0.000). Al utilizar el punto de corte de 30% se encontró diferencia significativa entre los dos grupos (Grupo 1: ≤ 30% y Grupo 2: > 30%) en la edad (39 años vs 44 años, P=0,000), en la concentración espermática (78,00 x 106/ml vs 36,75 x 106/ml, P=0,015), movilidad progresiva (40,85% vs 22,38%, P=0,000), morfología normal (12,0% vs 6,0%, P=0,004) y vitalidad espermática (85% vs 72,5%, P=0.000). También se demostró una correlación inversa entre el IFA y los parámetros de concentración (r=- 0,219 P=0,000), movilidad progresiva (r=-0,452 P=0,000), morfología normal (r=-0,322 P=0,000) y vitalidad (r=-0,452 P=0,000) en la población total. Se determino una correlación positiva significativa entre la edad y el IFA (0,267 P= 0.000). Conclusiones:Los resultados indican que el daño a nivel de ADN espermático en pacientes con muestra seminal alterado en algún parámetro es significativamente alto comparado al de los pacientes con muestra seminal normozoospermica. Se ha demostrado que los parámetros seminales (concentración, movilidad, morfología y vitalidad) están altamente correlacionados negativamente con el índice de fragmentación del ADN espermático. Se demostró una correlación positiva entre la edad y el IFA.
... Sperm DNA fragmentation is negatively correlated with ART outcomes and was identified as a major indicator of sperm quality [17-18, 24, 31, 34]. AR and mitochondrial biomarker MMP also can be regarded as predictors of semen quality in the general study population [35][36][37][38][39]. No difference was observed on the level of DFI, MMP and AR between these two techniques. ...
Backgroud: The aim of this study is to investigate the most effective and maneuverable technique for sperm preparation in conventional IVF cycles.
Method: A retrospective and laboratory-based study was conducted in patients who underwent their first cycle of IVF from January to December in 2016 to compare two sperm preparation techniques: direct swim-up without centrifugation (DSU) technique and density-gradient centrifugation followed by swim-up (DGC-SU) technique. A series of experiments in this study was designed to evaluate the efficiency of these two techniques which include: (i) assessment of quality and quantity of spermatozoa by comparing motility, DNA fragmentation index (DFI), acrosomal reaction (AR) and mitochondrial membrane potential (MMP) of DSU-separated sperm to DGC-SU-separated sperm, (ii) evaluation of safety of DSU technique by assessing the risks of bacterial contamination, (iii) analysis of feasibility of replacing DGC-SU with DSU technique by reviewing ART outcomes including fertilization rate, high-quality embryo rate, implantation rate, clinical pregnancy rate, take-home baby rate and abortion rate.
Results: Although there were no significant differences in DFI, AR and MMP between DSU-separated sperm and DGC-SU-separated sperm, significantly higher percentage of progressive motility in DSU-separated sperm were found than that in DGC-SU-separated sperm. Moreover, there were no significant differences between DSU and DGC-SU groups on ART outcomes based on data of fertilization rate, high-quality embryo rate, implantation rate, clinical pregnancy rate, baby delivery rate and abortion rate. In addition, no bacterial contaminations were found in culture medium samples of simulating fertilization from DSU group. However, it is noticeable that DSU technique required less time and labor for sperm preparation compared with DGC-SU.
... This is especially relevant if we consider that both IVF and ICSI bypass the sperm selection operating in vivo, increasing the risk of fertilizing the oocyte with defective spermatozoa that could lead to developmental failure and even affect the offspring in the long run (Fernandez-Gonzalez et al., 2008). This risk could be higher in the clinical practice since the incidence of sperm abnormalities, including DNA fragmentation, is higher in infertile men (Saleh et al., 2003;Ozmen et al., 2007;Schulte et al., 2010). ...
Almost 50% of the infertility cases are due to male factors. Assisted reproductive technologies (ARTs) allow to overcome the incapacity of these patients’ spermatozoa to fertilize the oocyte and produce a viable and healthy offspring, but the efficiency of the different techniques has still the potential to improve. According to the latest reports of the European Society of Human Reproduction and Embryology (ESHRE) and the Centers for Disease Control and Prevention of the United States (CDC), the percentages of deliveries per ART cycle in 2014 and 2016 were 21 and 22%, respectively. Among the reasons for this relatively low efficiency, the quality of the spermatozoa has been pointed out as critical, and the presence of high percentages of DNA-damaged spermatozoa in patients’ ejaculates is possibly one of the main factors reducing the ARTs outcomes. Thus, one of the main challenges in reproductive medicine is to ensure the highest quality of the spermatozoa used in ARTs, and specifically, in terms of genetic integrity. The latest techniques for the preparation and selection of human spermatozoa are herein discussed focusing on those proven to improve one or several of the following parameters: sperm genetic integrity, fertilization capacity, embryo production, and in vitro survival, as well as pregnancy and delivery rates following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In addition, we discuss the potential of techniques developed in non-human mammals that could be further transferred to the clinic.
... In fact, we also observed that in addition to ROS, hypertensive rats presented increased DNA fragmentation and number of spermatozoa with damaged acrosome. It was already demonstrated that reduction in testicular blood flow induced an increasing in DNA fragmentation in some germinative cells such as spermatogonia and primary spermatocytes 61 and damage of sperm DNA can occur at any step of spermatogenesis 63,64 . In other condition with vascular alterations, it was showed that patients with varicocele had reduced testicular arterial blood flow and also increased sperm DNA fragmentation 65,66 . ...
Arterial hypertension is a cardiovascular disease that leads to important systemic alterations and drastically impairs normal organ function over time. Hypertension affects around 700 million men of reproductive age and hypertensive men present increased risk for reproductive disorders, such as erectile dysfunction. However, the link between arterial hypertension and male reproductive disorders is associative at best. Moreover, many studies have reported associations between decreased male fertility and/or semen quality and alterations to general male health. In this study we aim to investigate the effect of systemic high blood pressure in sperm quality, sperm functional characteristics and testicular physiology in a rat model. Hypertensive rats presented altered testicular morphology – mainly vascular alterations and impaired testicular vasomotion. Hypertensive rats also presented decrease in sperm concentration, DNA integrity and increased percentages of sperm with dysfunctional mitochondria, intracellular superoxide anion activity and abnormal morphology. This study provides mechanistic insights by which arterial hypertension affects the testes, evidencing the testes as another target organ for hypertension as well as its impact on sperm quality.
... On the other hand, some authors [21,27,62,64,67] have indicated an obvious influence of sperm DNA damage (TUNEL, SCSA and SCD assay) on embryo development and pregnancy success achieved by spontaneous conception or medically assisted conception even when the percentage of sperm cells with non-mature chromatin was low (< 10-25%). Pregnancy loss occurred at < 10% sperm DNA fragmentation [68,69]. In addition, statistically significant correlations between sperm chromatin maturity and integrity with fertilization, embryo development and pregnancy results were noted. ...
Because sperm chromatin may play a key role in reproductive success, we verify the associations between sperm chromatin abnormalities, embryo development and the ability to achieve pregnancy. The evaluation of sperm chromatin maturity using aniline blue (AB), chromomycin A3 (CMA3) and toluidine blue (TB) staining were carried out in group of males from infertile couples that underwent ICSI. Low levels of sperm chromatin abnormalities (< 16%) were found in most subjects (> 50%). A higher percentage of TB-positive sperm cells were discovered in the men from couples who achieved ≤ 50% fertilized oocytes compared to men who achieved > 50%. No significant differences were discovered by the applied tests between the men from couples who achieved ≤ 50% and those who achieved > 50% high-quality embryos on the 3rd or 5th day after fertilization, nor between the men from couples who achieved pregnancy and those who failed. The sperm chromatin maturity did not correlate with the ICSI results. However, the ROC analysis revealed a significant predictive value of TB-positive spermatozoa only for fertilization. Therefore, the TB assay can be considered as a useful test for the prediction of fertilization. Our findings suggest that the level of sperm chromatin abnormalities of the examined men was not clinically significant. No found associations between sperm chromatin maturity and embryo development and the ability to achieve pregnancy. We could not exclude the effects of the repairing processes in the fertilized oocyte. The use of complementary tests that verify the status of the sperm chromatin seems justified.
... This is especially relevant in case of the intracytoplasmic sperm injection (ICSI) and could explains the low efficiency of ART in general [28]. Nearly half of the male patients 155 diagnosed as infertile show high levels of sperm DNA damage [29] and most patients subjected to fertility treatments show alterations in the sperm chromatin [30][31][32][33]. Moreover low sperm counts have been related to higher presence of chromosomal aberrations in the spermatozoa. ...
... This may be especially critical for ARTs and in particular for the technique of intracytoplasmic sperm injection used in fertility treatments, because of the risk of fertilizing the oocyte with spermatozoa with errors that could affect the offspring in the long run [64]. Indeed, in human clinical practice, this risk may be higher if we consider the higher incidence of DNA fragmentation in the spermatozoa of infertile men [65][66][67]. Accordingly, sperm selection prior to ART is becoming an important research field in which the main challenges are to discover the sperm subpopulation selected within the female genital tract and the mechanisms involved in their selection in vivo. Only after acquiring this knowledge will we be able to design efficient in vitro methods for selecting high-quality spermatozoa for use in ARTs [68]. ...
The mammalian oviduct is the place where life begins as it is the site of fertilization and preimplantation embryo development. Recent research has highlighted the important role played by the oviduct both in sperm selection for natural fertilization and in the genetic and epigenetic reprogramming of pre-implantation embryo development. This review examines oviduct fluid composition with a special emphasis on exosomes and the role played by the oviduct in sperm selection, early embryo development, and in reshaping the epigenetic landscape of the embryo. In addition, the implications of data obtained for improving assisted reproductive technologies are discussed.
... In the light of these findings, some authors have proposed that spermatozoa with DNA damage do not undergo physiological process such as capacitation and acrosome reaction. The scientific support for these notions is the negative correlation observed between DNA damage and acrosome reaction in human spermatozoa (Ozmen, 2007). Furthermore, Grunewald (2006) has shown that markers of apoptosis are not detectable in the capacitated and acrosome-reacted spermatozoa (Grunewald et al., 2006). ...
Sperm genomic integrity has a significant effect on intracytoplasmic sperm injection (ICSI) outcomes, especially post-implantation. Spermatozoa selected based on motility and morphology do not guarantee the genomic integrity of spermatozoa. Nearly fifty percentage of spermatozoa in infertile men with normal morphology present different degrees of DNA fragmentation. However, capacitated or hyperactivated spermatozoa show lower degrees of DNA fragmentation. Therefore, selection of hyperactivated spermatozoa may improve ICSI outcome. Routinely, for ICSI, fast-moving spermatozoa with A or B motility pattern are mainly selected for injection. The result of this study shows that in processed semen samples, hyperactivated spermatozoa are mainly observed in B motility pattern while, in viscous medium like polyvinylpyrrolidone (PVP), hyperactivated spermatozoa are mainly present in spermatozoa with C pattern of motility (nonprogressive). Therefore, we propose spermatozoa with C motility pattern which contains the main population of physiological or hyperactivated spermatozoa should be selected for ICSI.
... However, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Moreover, negative correlation was identified between DNA damage and acrosome reaction and/or viability of human spermatozoa (Ozmen et al., 2007). ...
... However, DNA damage should alter the special cellular functions of human spermatozoa, and lead to diminished acrosome reaction with reduced fertilization rates. Moreover, negative correlation was identified between DNA damage and acrosome reaction and/or viability of human spermatozoa (Ozmen et al., 2007). Recently, our group demonstrated that the specific SHBF pattern (total or partial) was not an efficacious means of selecting sperm chromatin packaging abnormalities, at least when observed by chromomycin A3 staining. ...
The application of assisted reproduction techniques has provided help to many men seeking to father a child, although the current success of these procedures remains suboptimal. Today some protocols allow sperm to be selected according to their ultrastructural morphology or surface molecular characteristics. On the other hand, successful human reproduction relies partly on the inherent integrity of sperm DNA. Therefore, it is now necessary to improve the safety of the sperm selection method. It is urgent to optimize procedures to isolate spermatozoa for ICSI with low risk of DNA damage. In recent years, two technologies have attracted the attention of specialists as methods capable of identifying a spermatozoon with low risk of DNA damage: Ultrastructural morphology sperm selection at high magnification and sperm head birefringence selection. This review analyses these two technologies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.
... In addition, DNA fragmentation is significantly more frequent in spermatozoa with total birefringence compared with those with partial birefringence (Petersen et al., 2011) that, according to this study centre's experience, are those having a reacted acrosome and a higher capacity of giving rise to implantation (Gianaroli et al., 2010). These findings altogether confirm what was already proposed by Ozmen et al. (2007); damage in DNA structure could negatively affect the sperm capacity to undergo the acrosome reaction and the consequent steps following its entry into the oocyte. This could explain the highest clinical outcome associated with the injection of reacted spermatozoa. ...
... DNA damage alters the special cellular functions of human spermatozoa, resulting in diminished acrosome reactions with reduced rates of fertilization. Ozmen et al. reported that negative correlations were identified between increased DNA damage and acrosome reactions and/or the viability of human spermatozoa, especially in cases involving reduced fertilization rates [61]. In addition, Morakinyo et al. reported that oxidative stress induced by calcium antagonists decreases the percentage value of acrosomal-reacted sperm in rats [62]. ...
We investigated sperm nuclear vacuolation in relation to acrosome reactions and the maintenance of sperm motility. Thirty male patients who visited our Male Infertility Clinic were enrolled. These patients underwent conventional semen analyses, Acrobeads tests, and high-magnification observation of the sperm head to evaluate the degree of nuclear vacuolation on the Acrobeads test scoring after 24 hours of incubation. The presence of acrosome reactions was evaluated using the Acrobeads test. The spermatozoa were classified into three groups: (I) those bound to MH61-beads, (II) motile spermatozoa that did not bind to MH61-beads, and (III) immotile spermatozoa that did not bind to MH61-beads. The percentage of spermatozoa with large nuclear vacuoles (%LNV) was compared between the three groups. The degree of sperm nuclear vacuolation was evaluated in 17,992 ejaculated spermatozoa. The mean %LNVs were 2.4% in group I, 5.8% in group II, and 9.8% in group III. These values were significantly different from each other (
P
<
0.001
, paired
t
-test). There were no correlations between the %LNV values and the Acrobeads scores. In conclusion, the degree of sperm nuclear vacuolation was significantly lower in the acrosome-reacted spermatozoa and spermatozoa with maintained motility, and higher in the immotile spermatozoa that did not bind to MH61-beads.
... 113, 114 DNA fragmentation may be an excellent marker of exposure to reproductive toxicants and a diagnostic tool for potential male infertility. [115][116][117] ...
Human semen is the end result of a sophisticated biological process hormonally regulated, produced by highly specialized cellular lines that differentiate in embryo but initiate division at puberty and will continue dividing throughout the entire life span of the individual in cycles of 72 days. Semen is a sensitive indicator of environmental, occupational and lifestyle exposures that may be altered by direct toxic effects and hormonal disruption. Damage may happen along the entire human life. However, while some exposures may produce reversible changes, others, especially damage to germinal cells in uterus or prepubertally may result in permanent sequelae. We review the main factors that affect human male fertility and their possible influence in current human reproduction. Some lifestyles, xenoestrogens, heavy metals and volatile organic compounds are already known to compromise reproductive male function. Nonetheless, many questions remain and we still know little about the effect of many other factors on male fertility.
... This hypothesis is confi rmed by various studies. Kasai et al. ( 2002 ) observed a direct association between motility, mitochondrial volume, and membrane potential within the sperm midpiece; mutations or deletions within mitochondrial DNA were shown to be associated with reduced sperm motility (Ozmen et al. 2007 ); and Speyer et al. ( 2010 ) noted a positive correlation between DFI and sperm midpiece. ...
The selection of spermatozoa without DNA fragmentation and chromosomal diseases prior to assisted reproductive techniques helps to optimize the outcome of the treatment; in particular, sperm selection prior to in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) is crucial. In fact, although ICSI has been successfully and safely applied worldwide for almost 20 years, at the present time we have no real knowledge regarding the hypothetical long-term side effects on ICSI adults, given the increased likelihood of spermatozoa with defective nuclear content fertilizing oocytes.
In the case of DNA damage, the basal sperm DNA fragmentation rate can be significantly reduced by some sperm processing procedures that improve the percentage of spermatozoa with normal chromatin structure by filtering out DNA-damaged spermatozoa. After this first step, new advances in micromanipulation can be performed to choose the “ideal” mature spermatozoa for ICSI, reducing potential damage to the gametes. In fact, it is possible to prevent fertilization by DNA-damaged and chromosomal-unbalanced spermatozoa by selecting ICSI sperm by maturation markers such as hyaluronic acid or other zona pellucida receptors. Furthermore, novel noninvasive imaging techniques can be valid tools for helping in the morphological selection of ICSI spermatozoa.
... It is well known that sperm DNA quality has been recognized as one of the most important markers of male reproductive potential [30,42,60], in contrast to standard semen parameters such as sperm density, motility and morphology that do not act as powerful discriminators between fertile and infertile men [14,21]. ...
Purpose:
An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia.
Methods:
In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period.
Results:
After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p<0.05), but we found a significant reduction in patients with high basal DFI values (>15 %), while no significant variation occurred in the patients with DFI values ≤ 15 %.
Conclusions:
Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 % .
The aim of our study was to evaluate the correlation between sperm quality and ploidy status of the derived blastocysts. We performed a retrospective analysis on a restricted pool of patients enrolling only those who had no female factors. Male patients with genetic factors affecting spermatogenesis were also excluded. We chose a maternal age ≤38 years to decrease the female factor, therefore the male factor was the main component of sterility. We divided the patients in four groups based on semen quality and comparing fertilization, pregnancy and euploidy rates above all. In total, 201 intracytoplasmic sperm injection (ICSI) cycles were enrolled in the study. Cycles were divided into four groups, according to semen source: normal semen, oligoasthenoteratozoospermia (OAT), cryptospermia or non-obstructive azoospermia (NOA). An extremely statistically lower fertilization rate was found in NOA patients. Unexpectedly, no differences were detected in blastocyst formation, euploidy, aneuploidy and mosaicism rates among the four groups. Interestingly, we also found a higher abortion rate comparing NOA to normal semen with an odds ratio of 4.67. In our study no statistically significant differences among the analyzed groups were found, showing little or no effect at all using spermatozoa from different semen sources or quality. This may be linked to the oocyte competence of fixing sperm DNA damage and it could be hypothesized that only sperm with a good rate of DNA integrity are able to fertilize the oocyte, explaining why poor quality semen is reflected in a low fertilization rate without effect on ploidy.
The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel-Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I 2 statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR = 0.74; 95% CI = 0.61-0.91; I 2 = 69) and pregnancy rate (RR = 0.83; 0.73-0.94; I 2 = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR = 0.78; 0.58-1.06; I 2 = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR = 0.68; 0.52-0.89; I 2 = 50) and pregnancy rates (RR = 0.72; 0.55-0.95; I 2 = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR = 0.48; 0.22-1.02; I 2 = 79). In the case of ICSI, non-significant trends were observed for implantation (RR = 0.79; 0.60-1.04; I 2 = 72) or pregnancy rates (RR = 0.89; 0.78-1.02; I 2 = 44), and live birth rate (RR = 0.92; 0.67-1.27; I 2 = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments.
This study was conducted to analyze the specific genes associated with sex-determination in Korean native cow. The highly organized spermatogenesis requires accurate spatial and temporal regulation of gene expression, which is governed by transcriptional, post-transcriptional, and epigenetic processes. Recently, farmers have been interested in determining the sexual identity of the calves in their farm. We analyzed the sperm of Korean native and Holstein cows, which were supplied from Hanwoo Improvement Center. We evaluated sperm motility and expression of sperm-specific genes after treating semen with both male- and female reagents. Sperm motility in Korean native cows decreased by approximately 10% in the first 30 minutes after treatment with sex-determination reagent. However, sperm motility of Holstein cows decreased to 60-70% after 15 minutes and to 20-30% after 30 minutes. We selected six specific genes expressing in the spermatozoa to analysis the gene expression level. The Real-time PCR results suggest that the selected genes (Gimap4, Tmeff1, Rac2, Abi2, Rac1, and Clu) were highly expressed in the group treated with the male reagent compared to the group treated the female reagent and to the untreated-group (control). In the present study, we suggest that the selected genes play a pivotal role in sex-determination.
DNA fragmentation, or the accumulation of single- and double-strand DNA breaks, is a common property of sperm, and an increase in the level of sperm DNA fragmentation is known to influence natural reproduction. The effect of sperm DNA fragmentation on male infertility and assisted reproductive treatment (ART) outcomes remains controversial and is one of the most frequently debated topics of reproductive medicine. For the past 30 years, a number of assays have been developed to quantify the level of sperm DNA fragmentation. In this chapter, we review the causes of sperm DNA fragmentation, describe the commonly used tests to evaluate these abnormalities, and perform a systematic review of existing studies to determine the impact of sperm DNA fragmentation on male fertility and ART outcomes.
Progesterone (P4) is crucial for the physiological function of spermatozoa. In the study, we investigated the correlation between P4‐induced sperm acrosome reaction (AR) and parameters including sperm progressive motility, normal morphology and sperm DNA fragmentation (SDF), and compared the in vitro fertilization (IVF) predictive values of these indicators based on the multivariate regressions analysis and receiver operator characteristics (ROC) curve analyses. The results demonstrated a negative correlation between P4‐induced sperm AR and the SDF, with the correlation −9.05 (−17.25 to −0.84), p<0.05, n = 47). No relationship was found between the sperm progressive motility, normal morphology and the induced AR. The P4‐induced AR and SDF were both significantly correlated to the fertilization rate. ROC curve analyses indicated that P4‐induced AR was a better prognostic predictor for the fertilization rate compared with the SDF, with the areas under the curve 0.729 (0.580–0.849), p<0.01 and 0.637 (0.484–0.772), p=0.16 respectively. The cut‐off value for P4‐induced AR to predict “50% fertilization rate” was 23.4% with sensitivity and specificity of 63.3% and 88.2% respectively. The overall results indicated that the assessment of P4‐induced AR seemed to be a more sensitive indicator for fertilization rate in vitro compared with other sperm parameters.
Acridine orange (AO) staining is one of the methods used to diagnose the DNA fragmentation status in sperm cells. Interferometric phase microscopy (IPM) is a an optical imaging method based on digital holographic microscopy that provides quantitative morphological and refractive index imaging of cells in vitro without the need for staining. We have imaged sperm cells using stain‐free IPM in order to estimate different cellular parameters, such as acrosome dry mass and size, in addition to an embryologist evaluation according to the WHO‐2010 criteria. Following this, the same sperm cells were stained by AO, imaged using a fluorescence confocal microscope and assessed by the AO‐emitted color, forming five DNA fragmentation groups. These DNA fragmentation groups were correlated with the embryologist‐based classification and the IPM‐based morphological parameters. Our results indicate on significant differences in IPM‐based parameters between groups with different fragmentation levels. Specifically, the size of the acrosome, as measured from stain‐free IPM, is a good predictor for the presence of intact DNA. Based on the validation with AO, we conclude that stain‐free IPM images analyzed digitally may assist in selecting sperm cells with intact DNA prior to intracytoplasmic sperm injection (ICSI). This information may potentially increase percentage of successful pregnancies.
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The integrity and stability of the sperm genome is protected by DNA repair mechanisms both in the male germ line cells and oocyte. This chapter focuses on our understanding of DNA repair mechanisms in the oocyte and zygote after fertilization. The chapter also reviews the origin of DNA damage, the effect of sperm DNA damage on reproductive outcomes and sperm selection methods in assisted reproductive techniques.
Sperm DNA fragmentation has been associated with reduced fertilization rates, embryo quality, pregnancy rates and increased miscarriage rates. Various methods exist to test sperm DNA fragmentation such as the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion (SCD) test, the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay and the single cell gel electrophoresis (Comet) assay. We performed a systematic review and meta-analysis to assess the value of measuring sperm DNA fragmentation in predicting chance of ongoing pregnancy with IVF or ICSI. Out of 658 unique studies, 30 had extractable data and were thus included in the meta-analysis. Overall, the sperm DNA fragmentation tests had a reasonable to good sensitivity. A wide variety of other factors may also affect the IVF/ICSI outcome, reflected by limited to very low specificity. The constructed hierarchical summary receiver operating characteristic (HSROC) curve indicated a fair discriminatory capacity of the TUNEL assay (area under the curve (AUC) of 0.71; 95% CI 0.66 to 0.74) and Comet assay (AUC of 0.73; 95% CI 0.19 to 0.97). The SCSA and the SCD test had poor predictive capacity. Importantly, for the TUNEL assay, SCD test and Comet assay, meta-regression showed no differences in predictive value between IVF and ICSI. For the SCSA meta-regression indicated the predictive values for IVF and ICSI were different. The present review suggests that current sperm DNA fragmentation tests have limited capacity to predict the chance of pregnancy in the context of MAR. Furthermore, sperm DNA fragmentation tests have little or no difference in predictive value between IVF and ICSI. At this moment, there is insufficient evidence to recommend the routine use of sperm DNA fragmentation tests in couples undergoing MAR both for the prediction of pregnancy and for the choice of treatment. Given the significant limitations of the evidence and the methodological weakness and design of the included studies, we do urge for further research on the predictive value of sperm DNA fragmentation for the chance of pregnancy after MAR, also in comparison with other predictors of pregnancy after MAR.
Sperm DNA damage is prevalent among infertile men and is known to influence natural reproduction. However, the impact of sperm DNA damage on assisted reproduction outcomes remains controversial. Here, we conducted a meta-analysis of studies on sperm DNA damage (assessed by SCSA, TUNEL, SCD, or Comet assay) and clinical pregnancy after IVF and/or ICSI treatment from MEDLINE, EMBASE, and PUBMED database searches for this analysis. We identified 41 articles (with a total of 56 studies) including 16 IVF studies, 24 ICSI studies, and 16 mixed (IVF + ICSI) studies. These studies measured DNA damage (by one of four assays: 23 SCSA, 18 TUNEL, 8 SCD, and 7 Comet) and included a total of 8068 treatment cycles (3734 IVF, 2282 ICSI, and 2052 mixed IVF + ICSI). The combined OR of 1.68 (95% CI: 1.49-1.89; P < 0.0001) indicates that sperm DNA damage affects clinical pregnancy following IVF and/or ICSI treatment. In addition, the combined OR estimates of IVF (16 estimates, OR = 1.65; 95% CI: 1.34-2.04; P < 0.0001), ICSI (24 estimates, OR = 1.31; 95% CI: 1.08-1.59; P = 0.0068), and mixed IVF + ICSI studies (16 estimates, OR = 2.37; 95% CI: 1.89-2.97; P < 0.0001) were also statistically significant. There is sufficient evidence in the existing literature suggesting that sperm DNA damage has a negative effect on clinical pregnancy following IVF and/or ICSI treatment.
The selection of the individual spermatozoon in ICSI is routinely performed by the observation of its motility and morphology. However, in case of severe oligoasthenozoospermia or non-obstructive azoospermia needing the use of testicular sperm, other methods are necessary to help the embryologist making this choice. According to some authors, sperm processing before ICSI seems to limit the DNA fragmentation index, and in this way improve ICSI outcomes. Moreover, IMSI is potentially a good option in some specific indications such as severe teratozoospermia, or repeated ICSI failures. Other methods based on sperm structure, as sperm head birefringence observation, or based on its function, like the hyaluronic acid or zona pellucida binding capacity, could be of interest, but still need to be confirmed. Finally, in case of akinetozoospermia, the use of functional tests, such as pentoxifylline test, HOS-test, or to a lesser extent laser touch, makes the selection of viable spermatozoa easier. Nevertheless, studies on larger series have to be conducted to evaluate and precise the interest of each of these methods and their indications, before considering an application on larger scale.
Objective:
To examine whether sperm DNA fragmentation has an effect on pregnancy and miscarriage after IVF and/or intracytoplasmic sperm injection (ICSI).
Design:
Systematic review and meta-analysis.
Setting:
University-affiliated teaching hospital.
Patient(s):
Infertility patient(s).
Intervention(s):
An exhaustive electronic literature search was conducted on MEDLINE, Google Scholar, and the Cochrane Library, from database inception to October 2013. We included clinical trials that examined the influence of sperm DNA damage on pregnancy and miscarriage of IVF/ICSI.
Main outcome measure(s):
The outcomes of interest were pregnancy rate and miscarriage rate.
Result(s):
In the analysis of pregnancy, 16 cohort studies (3,106 couples) were included. Of these, 14 studies (2,756 couples, 965 pregnancies) that also mentioned miscarriage were identified in the analysis of miscarriage. Meta-analysis showed that high-level sperm DNA fragmentation has a detrimental effect on outcome of IVF/ICSI, with decreased pregnancy rate and increased miscarriage rate. The stratified analysis by type of procedure (IVF vs. ICSI) indicated that high sperm DNA damage was related to lower pregnancy rates in IVF but not in ICSI cycles, whereas it was associated with higher miscarriage rates in both IVF and ICSI cycles.
Conclusion(s):
The results indicate that assays detecting sperm DNA damage should be recommended to those suffering from recurrent failure to achieve pregnancy. Selection of sperm without DNA damage for use may improve the clinical outcome of ART. The data also provide a rationale for conducting further research aimed at evaluating the underlying mechanism(s) responsible for the detrimental effect of high sperm DNA fragmentation and the potential therapy.
The evaluation of the morphology of human spermatozoa varies widely between and sometimes even within laboratories. The purpose of this study was to determine whether the method that has been developed in our laboratory and which resulted in the use of stricter criteria for the evaluation of sperm morphology is a practical, reliable and repeatable method and to establish the within and between observer variations. The criteria used for a 'normal' spermatozoon are based on the appearance of spermatozoa found in the mucus of the upper endocervical canal. The results of the morphological evaluations of 26 samples by four observers were statistically analysed by various methods. The method of Barnett showed a high degree of relative accuracy between observers with error variances of between 2.89 and 19.67 as well as high Spearman rank correlation coefficients of between 0.8675 and 0.6537 (P less than 0.0003). The Spearman correlation coefficient for 15 duplicate evaluations by one observer was 0.9650 (P less than 0.0001) while the coefficients of variation for repeated evaluations of single samples were also within acceptable limits. Based on these results, the method described in this article allows comparable and reliable results between and within observers to be obtained. From this and other studies it can be concluded that the method also has a good prognostic value for the prediction of expected IVF fertilization, the hamster test and hemizona assay.
We are in the process of assessing the response of cancer tissues to chemotherapy, evaluating, among other points, the proportion of cancer cells undergoing apoptosis. However, the apoptotic index obtained with the original TUNEL technique was lower than that obtained by evaluation of apoptosis on H&E-stained sections. Here we describe a small modification of the TUNEL technique that significantly increases the sensitivity of the assay. In the nonmodified TUNEL technique, a digoxigenin-labeled probe is detected using a direct peroxidase-conjugated system, whereas here we report the advantage of using a streptavidin-biotin-immunoperoxidase system. This, in conjunction with pretreatment of tissue sections with proteinase K and microwave irradiation, improved the detection of apoptotic cells.
The ability of human embryos to undergo normal development has been shown previously to be subject to strong paternal (sperm-derived) effects. This study was undertaken to determine whether paternal influences on human embryo quality are detectable as early as the first cell cycle after fertilization.
The quality of zygotes and cleaving embryos resulting from sibling donor oocytes fertilized by sperm from different patients were compared in a donor oocyte-sharing programme.
Fertilizations with sperm from certain individuals repeatedly resulted in the formation of high proportions of zygotes with abnormal pronuclear morphology that subsequently tended to cleave slowly and to show extensive fragmentation and blastomere irregularities. This phenomenon was observed with oocytes from two different donors for each of these individuals and contrasted with normal developmental performance of embryos resulting from sibling oocytes fertilized by sperm from other men with similar basic sperm characteristics. Fertilization rates were not related to these differences.
These data point to a very early onset of paternal effects that condition human embryo development. These effects may be both of genetic (related to the minor gene activity of the male pronucleus) or epigenetic (related to the sperm-derived oocyte-activating factor or sperm centrosome) origin.
We aimed to investigate whether sperm DNA quality may predict intrauterine insemination (IUI) outcome.
The study was designed in a prospective cohort fashion, at a tertiary centre for reproductive medicine. A total of 119 patients underwent 154 cycles of IUI. Parameters related to demography, cycle management and semen sample used for IUI were evaluated. Conventional semen parameters, morphology (strict criteria), sperm DNA fragmentation and stability [evaluated by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and acridine orange staining under both acid and acid + heat denaturing conditions respectively] were measured. The main outcome measure was clinical pregnancy, defined as ultrasonographic visualization of intrauterine gestational sac(s).
Logistic regression analyses were done on six sets of data, including all cycles combined, cycles with washed samples, first cycle of each couple, first cycle of each couple with washed samples, cycles stimulated with gonadotrophins and finally gonadotrophin-stimulated cycles with washed samples. The number of pre-ovulatory follicles on day of hCG, the age of the woman and the percentage of sperm with acid- + heat-resistant DNA were the parameters that predicted IUI outcome in most of these data subsets. For the gonadotrophin-stimulated cycles, age of the man appeared as a predictor as opposed to that of the woman; and for the cycles within this subgroup, where the semen sample was washed, sperm DNA fragmentation and age of the man were the only two parameters to predict IUI outcome. No samples with >12% of sperm having DNA fragmentation resulted in pregnancy.
The number of follicles, age of the woman/man and sperm DNA quality may predict IUI outcome.
This prospective study compared the acrosome reaction following ionophore challenge (ARIC) versus conventional sperm parameters and sperm velocities in predicting successful outcome following ovarian stimulation and intrauterine insemination.
All patients were offered a maximum of three treatment cycles. Conventional semen analysis was performed and sperm velocities were measured using computer-aided sperm analysis. Acrosome-reacted sperm were stained using chlortetracycline after ionophore challenge. Multiple logistic regression analysis and the receiver-operator characteristic curve analysis were applied to determine the best predictive variables and their cut-off values.
ARIC score was the most significant variable in predicting pregnancy, followed by the percentage of induced acrosome-reacted sperm, serum estradiol levels on the day of hCG and sperm morphology by strict criteria. Higher spontaneous acrosome reaction had a negative relationship with pregnancy. ARIC score of 10% had a sensitivity of 85.3% and a specificity of 85.5%. The positive and negative predictive values were 64.2 and 96.6% respectively and the false positive and negative rates were 14.7 and 14.5% respectively.
ARIC score was a better predictor of pregnancy than conventional sperm parameters and sperm velocities.
Standard sperm characteristics are poor predictors of the outcome of IVF treatments. On the contrary, sperm genome quality has been emphasized for several years as playing a major role in early embryogenesis, thus in the success of IVF attempt.
Sperm DNA fragmentation from a selected group of 104 couples undergoing assisted reproductive techniques (ART) (IVF: n = 50; and ICSI: n = 54) was measured by TUNEL assay and correlated with semen and ART outcomes.
A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >10%, a significant decrease of the fertilization rate was observed. No correlation was found between sperm DNA fragmentation and embryo quality. A high proportion of sperm with fragmented DNA was a pejorative factor to obtain pregnancies when ICSI was performed, but there was no relationship when conventional IVF was performed.
The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of ICSI outcome, whereas embryo quality based on morphological criteria, appeared unaffected by DNA fragmentation.
Sperm DNA integrity is essential for the accurate transmission of genetic information. The clinical significance of this assessment lies in its association with not only natural conception rates, but also the success of assisted reproduction technology (ART). It has been reported that sperm chromatin structure assay (SCSA) identified thresholds for negative pregnancy outcome after ART when the DNA fragmentation index (DFI), previously known as COMPalphat, was >30%.
In a prospective clinical study, we examined 34 male infertile patients, the husbands of women undergoing conventional IVF or ICSI. SCSA and ART were carried out on semen aliquots taken from the same ejaculate. Fertilization rate, embryo quality and pregnancy rates were correlated to SCSA parameters, DFI and highly DNA stainable (HDS) cells.
No differences were seen in SCSA parameter values between patients initiating pregnancies and not doing so in either ICSI or conventional IVF. Pregnancies and normal delivery were obtained even with high levels of DFI.
There is still controversy over whether analytical techniques currently in use are able to identify the level of damage to spermatozoa. Large-scale studies should be conducted in different clinical settings to determine the effects of sperm DNA damage on the outcome of ART.
The extent of sperm DNA fragmentation, which can be measured by the TUNEL assay, is one of the determinants of male fertility. However, the clinical application of this test to in-vivo situations is difficult owing to the absence of a statistically validated threshold value.
The aim of this study was to compare the results of TUNEL assay applied to semen samples from men of proven fertility (n = 47) and patients from an infertile population (n = 66), in order to establish a discriminating threshold value.
Infertile patients had a higher mean level of DNA fragmentation than men of proven fertility (40.9 +/- 14.3% versus 13.1 +/- 7.3%, respectively; P < 0.001). The area under the receiver operating characteristics curve was 0.93 for 20% sperm DNA fragmentation. The calculated threshold value for TUNEL assay to distinguish between fertile controls and infertile men was 20%. At this threshold, specificity was 89.4 [95% confidence interval (CI) 83.7-95.1] and sensitivity was 96.9% (95% CI 93.8-100). The positive and negative predictive values of the 20% sperm DNA fragmentation threshold were high: 92.8% (95% CI 87.9-97.5) and 95.5% (95% CI 91.6-99.3), respectively.
This study demonstrates that sperm DNA fragmentation, as measured by TUNEL assay, is a highly valuable indicator of male fertility.
Sperm are a highly specialized cell type derived to deliver the paternal haploid genome to the oocyte. The epigenetic, or gene regulatory, properties and mechanisms of the sperm assist in preparation of the paternal genome to contribute to embryogenesis and the genome of the zygote. Many recent studies have addressed the issue of altered epigenetic processes in the sperm. This review evaluates the current understanding of DNA damage, chromosome aneuploidy, reduced telomere length, malformations of the centrosome, genomic imprinting errors, altered mRNA profiles, and abnormal nuclear packaging in the sperm prior to fertilization and the observed effects on embryogenesis. Attention has also been given to understanding the underlying etiology of sperm with altered epigenetic mechanisms in humans.
Objective: To determine the incidence of DNA fragmentation in human sperm used for intracytoplasmic sperm injection (ICSI) and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in ICSI.Design: Descriptive and correlational clinical study.Setting: Tertiary care fertility clinic.Patient(s): A total of 150 semen samples was collected from men in the ICSI program.Intervention(s): For each sample, sperm wash and swim-up were performed, and the percentage of recovered sperm with DNA fragmentation was determined with the use of terminal transferase-mediated deoxyuridine triphosphate-biotin end labeling.Main Outcome Measure(s): The percentage of sperm with DNA fragmentation was correlated with semen analysis parameters and ICSI fertilization rates.Result(s): The mean (±SD) percentage of sperm with fragmented DNA was 14.5% ± 1.5% and ranged from 0.5% to 75%. A significant negative association was found between the percentage of sperm with DNA fragmentation and the ICSI fertilization rate. We also observed that the motility and morphology of the ejaculated sperm were correlated negatively with the percentage of DNA fragmentation in the washed sperm recovered by the swim-up technique.Conclusion(s): Our results suggest that when poor-quality semen samples are used for ICSI, there is a greater likelihood that some sperm selected for injection, despite appearing normal, contain fragmented DNA. Whether sperm DNA damage may contribute to failure of pronuclear formation and embryo development in some apparently unfertilized ICSI oocytes is unclear.
We describe two methods for detecting acrosome reactions of human sperm at the light microscopic level. The techniques include the use of a supravital stain to detect dead sperm in order to differentiate between “physiological” and “degenerative” acrosome reactions. Sperm are incubated with the supravital stain Hoechst 33258 (a fluorescent DNA-binding dye with limited membrane permeability), washed, suspended in 95% ethanol for fixation and permeabilization, and dried onto slides. The sperm are then labeled either by indirect immunofluorescence using rabbit anti-human sperm antiserum or with fluoresceinated Pisum sativum agglutinin (PSA). Both probes intensely label the acrosomal region of acrosome-intact sperm. Electron microscopy revealed the major site of PSA binding to be the acrosomal contents. Acrosome-reacted sperm have diminished acrosomal labeling by both probes; sperm with nuclei labeled by Hoechst stain are considered nonviable, and are excluded from the assay. Both assays are rapid, give similar results, and detect an increase in acrosome reactions following exposure to the ionophore A23187.
To determine the incidence of DNA fragmentation in human sperm used for intracytoplasmic sperm injection (ICSI) and to correlate any detected DNA damage with semen analysis parameters and fertilization rates in ICSI.
Descriptive and correlational clinical study.
Tertiary care fertility clinic.
A total of 150 semen samples was collected from men in the ICSI program.
For each sample, sperm wash and swim-up were performed, and the percentage of recovered sperm with DNA fragmentation was determined with the use of terminal transferase-mediated deoxyuridine triphosphate-biotin end labeling.
The percentage of sperm with DNA fragmentation was correlated with semen analysis parameters and ICSI fertilization rates.
The mean (+/- SD) percentage of sperm with fragmented DNA was 14.5% +/- 1.5% and ranged from 0.5% to 75%. A significant negative association was found between the percentage of sperm with DNA fragmentation and the ICSI fertilization rate. We also observed that the motility and morphology of the ejaculated sperm were correlated negatively with the percentage of DNA fragmentation in the washed sperm recovered by the swim-up technique.
Our results suggest that when poor-quality semen samples are used for ICSI, there is a greater likelihood that some sperm selected for injection, despite appearing normal, contain fragmented DNA. Whether sperm DNA damage may contribute to failure of pronuclear formation and embryo development in some apparently unfertilized ICSI oocytes is unclear.
In order to achieve a clinical pregnancy rate higher than that achieved following initial adoption of in-vitro fertilization embryo transfers, more than one embryo is transferred. This has led to a substantial increase in unwanted multiple pregnancy rates with IVF as compared with natural conception. What is therefore required is a simple, clinically useful embryo scoring system, to reflect embryo developmental potential, which will enable the selection of the optimal number of embryos to transfer in order to achieve the maximum pregnancy rate with a low incidence of high order multiple pregnancies. We believe that the Cumulative Embryo Score (CES) achieves these aims. On the day of embryo transfer the grade of each embryo transferred was multiplied by the number of blastomeres to produce a score for each embryo, and summation of the scores obtained for all the embryos transferred gave the CES. The grouped pregnancy rates obtained rose as the CES increased to maximum of 42. A continued increase in the CES above 42 did not result in any further rise in the pregnancy rate. However, an analysis of all our IVF pregnancies showed that the multiple pregnancy rate continued to rise above a CES of 42. By restricting the CES per embryo transfer to 42, 78% of triplet pregnancies and 100% of the quadruplet IVF pregnancies could have been predicted and potentially avoided.
A full understanding of the acrosome reaction is central to understanding sperm function. Acrosomal status can be determined on living, motile sperm in only a few mammalian species. For other species, many light microscopic methods have been developed, including colored stains for bright-field microscopy, and probes for fluorescence microscopy. We review the existing methods and the criteria that should be considered in the choice of an assay.
The purpose of this study was to examine the correlation between DNA strand breaks in human spermatozoa and semen quality, fertilization rate and IVF outcome.
A total of 50 men suffering from unexplained infertility and 50 men with oligozoospermia undergoing IVF treatment entered a prospective study. Sperm samples were assessed according to the WHO manual and for the presence of DNA strand breaks in spermatozoa. The study was blinded for the technician involved in assessment of DNA strand breaks. IVF was carried out according to a long down regulation protocol using GnRH, FSH and hCG. The ova were inseminated with 200,000 spermatozoa/ml. Embryos were transferred on day 2 after fertilization with a maximum of three embryos.
This study demonstrates a negative correlation between the proportion of spermatozoa having DNA strand breaks and the proportion of oocytes fertilized after IVF.
The number of human spermatozoa with DNA strand breaks is a good predictor for fertilization rate in couples suffering from unexplained infertility and undergoing IVF treatment.
To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears.
The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined.
Clinical and academic research environment.
59 patients undergoing fertility treatment.
Sperm samples leftover from IVF procedures were fixed and processed for the comet assay.
Sperm head DNA density and sperm variables.
A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology.
The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.
The acrosome reaction, which is essential for fertilization, includes fusion and vesiculation of the plasma membrane with the outer acrosomal membrane of spermatozoa, thereby releasing the acrosomal content. Determination of the ability of spermatozoa to undergo the acrosome reaction has proved to be a useful parameter in evaluation of infertile patients. The objective of this study was to compare cytochemical techniques, such as double stain (Giemsa/trypan blue) and triple stain (Bismarck brown/rose bengal/trypan blue), with a fluorescence method using Pisum sativum agglutinin fluorescein conjugate and Hoechst dye N degrees 33258 (double fluorescence). Whereas the cytochemical methods are easy to perform in general laboratories, the fluorescence technique requires special and costly instrumentation. In semen obtained from fertile donors, spermatozoa were selected by the swim-up technique and the acrosome reaction was induced by incubation at low temperature. The percentages of vital and acrosome-reacted spermatozoa were determined after incubation at 4 degrees C and at room temperature. No statistically significant difference was found between double fluorescence (viability 86.3%, acrosome reaction 14.7%) and triple stain (viability 85.3%, acrosome reaction 17%) (P > 0.05). On the other hand, the double stain technique showed different values for viability (70.3%) and acrosome reaction (42.5%) (P < 0.05). In conclusion, triple stain yielded results similar to those obtained by the fluorescence technique in evaluating the acrosome reaction and can therefore easily be used in general or research laboratories.
To evaluate the usefulness of the Hoechst 33258/FITC-Pisum sativum agglutinin (FITC-PSA) staining for simultaneous assessment of viability and acrosome reaction rate (%AR) of human sperm.
Fresh sperm was collected 13 fertile donors provided fresh semen. We used motile sperm selected by the "swim-up" procedure using modified HTF. Hoechst 33258 was added and co-incubated with sperm for 10 min. Samples were washed free of unbound stain and the sperm were mounted as smears on glass slides. After drying, sperms were incubated with FITC-PSA for 30 min. Sperm were examined by fluorescence microscopy. Also, FITC-Concanavalin A (FITC-ConA) staining and vital staining with yellowish eosin were performed simultaneously. The correlation of viability and %AR were analyzed.
Four different staining patterns were observed and clearly distinguished as follows: a) Viable acrosome-reacted sperm, b) Viable acrosome-intact sperm, c) dead acrosome-reacted sperm, d) dead acrosome-intact sperm. There was significant correlation between the results obtained by Hoechst 33258 and vital staining methods in viability of human sperm (r = 0.927, P < 0.001). There was significant correlation between the two methods (FITC-PSA and FITC-ConA) in %AR of human sperm (r = 0.92, P < 0.001).
Viability and acrosome status of a human sperm can be easily assessed simultaneously by Hoechst 33258/FITC-PSA staining method. This combination method is considered useful to evaluate sperm function.
Previous studies have indicated that sperm quality may be related to unexplained recurrent pregnancy loss. This study evaluated the degree of sperm DNA fragmentation using the TUNEL assay on sperm from 24 couples with unexplained recurrent pregnancy loss (RPL) compared to sperm from 2 control groups: donors of known fertility and unscreened men from the general population. The percentage of sperm staining positive for DNA fragmentation was increased (p < .001) in the RPL group (38 +/- 4.2) compared to the donor (11.9 +/- 1.0) or general population (22 +/- 2.0) control groups. In the RPL group, no correlation was observed between semen quality parameters and the TUNEL data. These data indicate that some RPL patients have a significant increase of sperm DNA fragmentation, which may be causative of pregnancy loss in some patients.
Despite the ever-increasing knowledge of the fertilization process, there is still a need for better understanding of the causes of sperm DNA fragmentation and its impact on fertilization and pregnancy. For this reason, human sperm DNA fragmentation was investigated by means of the terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and the production of reactive oxygen species (ROS) in the ejaculate and in the spermatozoa themselves. These data were correlated with fertilization and pregnancy data from IVF and intracytoplasmic sperm injection (ICSI) patients. Sperm DNA fragmentation did not correlate with fertilization rate, but there was a significantly reduced pregnancy rate in IVF patients inseminated with TUNEL-positive spermatozoa. ICSI patients exhibited the same tendency. This implies that spermatozoa with damaged DNA are able to fertilize an oocyte, but at the time the paternal genome is switched on, further development stops. The determination of ROS in the ejaculate and the percentage of ROS-producing spermatozoa revealed markedly stronger correlations between sperm functions (i.e. motility) and the percentage of ROS-producing spermatozoa. The influence of seminal leukocytes, known to produce large amounts of oxidants, on sperm DNA fragmentation should not be neglected.
Sperm concentration and motility are poor predictors of the outcome of intrauterine insemination (IUI), hysteroscopic intratubal insemination (HIT), or complete fertilization failure (CFF) in conventional IVF. We investigated whether the calcium ionophore-induced acrosome reaction (AR) constitutes an additional indicator of CFF and pregnancy that is independent of these semen parameters.
Infertile couples with no female factor (n=388) and women with tubal obstruction (n=32) were studied: IVF (n=133), ICSI (n=72), HIT (n=245) and IUI (n=61). The percentage of acrosome-reacted sperm in relation to viable sperm was calculated. Receiver operating characteristic curve and multiple logistic regression analyses were used to determine threshold values and the best predictor for CFF and pregnancy.
Threshold values of AR for predicting CFF in IVF and pregnancy in IVF and HIT + IUI were 21, 26 and 22% respectively. These values were independent of the conventional semen analysis parameters. CFF was lower (2 versus 20%; P<0.01) and the pregnancy rate was higher (46 versus 24% P<0.05) for those with AR >21% in IVF. CFF and pregnancy rate in ICSI did not differ according to AR. Pregnancy rate was higher for those with an AR >22% for HIT + IUI (23 versus 11% P<0.01).
Ionophore-induced AR appears to be a useful indicator in addition to routine semen analysis for selection of patients for treatment with appropriate assisted reproduction procedure.
To evaluate sperm DNA fragmentation in correlation with sperm parameters and IVF/intracytoplasmic sperm injection (ICSI) outcomes.
Retrospective review.
A tertiary infertility referral clinic.
We collected 303 semen samples from patients undergoing IVF with or without ICSI.
Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program.
The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes.
Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was >10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes.
The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates.
The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.
Previous studies have shown that repeated intracytoplasmic sperm injection (ICSI) failures can be caused by a paternal effect. Other studies have suggested that ICSI results are compromised if morphologically abnormal spermatozoa are injected into oocytes. This study was undertaken to evaluate the usefulness of a high-magnification optical system to select spermatozoa to be used for ICSI (high-magnification ICSI) in couples with repeated conventional ICSI failures. Couples with two or more previous conventional ICSI failures underwent an additional conventional ICSI attempt, followed by a high-magnification ICSI attempt. The outcomes of the two sequential attempts were compared. In 72 of these patients, sperm DNA integrity was assessed. In the whole group of 125 couples with repeated ICSI failures, high-magnification ICSI improved clinical outcomes (pregnancy, implantation, delivery and birth rates) without affecting biological outcomes (fertilization and cleavage rates, embryo morphology). The improvement of clinical ICSI outcomes was evident both in patients with an elevated degree of sperm DNA fragmentation and in those with normal sperm DNA status. It is concluded that high-magnification ICSI improves clinical outcomes in couples with previous repeated conventional ICSI failures.
Meta-analyses were conducted to investigate the relationship of sperm DNA fragmentation on pregnancy outcome using in-vivo fertilization, IUI, routine IVF and ICSI. Couples with no known infertility problems were 7.0 times (CI 3.17, 17.7) more likely to achieve a pregnancy/delivery if the DNA fragmentation index (DFI) was <30% (n = 362, P = 0.0001) using in-vivo fertilization. Infertile couples using IUI were 7.3 times (CI 2.88, 18.3) more likely to achieve a pregnancy/delivery if their DFI was <30% (n = 518, P = 0.0001). With routine IVF, infertile couples were approximately 2.0 times (CI 1.02, 2.84) more likely to become pregnant if their DFI was <30% (n = 381, P = 0.03). For ICSI and/or routine IVF, the results showed a non-significant trend where infertile couples were 1.6 times (CI 0.92, 2.94) more likely to achieve a pregnancy/delivery if the DFI was <30% (n = 323,P = 0.06). The in-vivo and IUI meta-analyses were similar, indicating that IUI infertility patients with <30% DFI have as good a statistical probability of obtaining a pregnancy/delivery as in-vivo presumably fertile couples with the same DFI. These meta-analyses show that the Sperm Chromatin Structure Assay infertility test was significantly predictive for reduced pregnancy success using in-vivo, IUI and routine IVF, and to a lesser extent ICSI fertilization.
The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer program
- R Steer
- Cj Mills
- Sl
- Tan
Steer R, CJ Mills, SL Tan et al. 1992 The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in-vitro fertilization and embryo transfer program. Human Reproduction 7, 117–119.
DNA strand breaks in human spermatozoa: correlation with fertilization in vitro in oligozoospermic men and in men with unexplained infertility
- Host
Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection
- Lopes